1. Field of the Invention
The invention pertains to artificial collagen oligopeptides and related compounds useful in clinical (including veterinary) applications.
Proteins and oligopeptide fragments thereof derived from animal sources have numerous well-known clinical uses. Collagen in particular is useful in the production of matrices for the support of various types of cells to promote the regeneration of damaged tissue such as nerve, bone, skin, or vascular tissues, and as a component of fibrin sealants. Typically, clinical grade commercial proteins such as collagen are isolated from their mammalian source, purified and sterilized to the extent feasible without denaturing the molecule, and, often, chemically modified to specialized use.
While natural collagen has many clinical advantages for such applications, including biocompatibility, shapability, and hemostatic properties, the use of mammal (including human- and bovine-)derived collagen presents potential hazards, especially the transmission of infectious agents from donor to donee. For example, the safety of collagen of animal origin has recently been placed in doubt with the recognition of the prevalence in Europe of bovine spongious encephalitis. This disease of unclear causes is of great concern for collagen users, since most of commercial collagen is of bovine origin and efficacy of conventional purification and sterilization procedures against the causative pathogen(s) is presently purely hypothetical. Further, the use of collagen of human origin has not obviated problems of possible contagion. Even though collagen purification methods currently in use appear to effectively eliminate viruses and retroviruses present, the possibility of the presence of other infectious agents unsusceptible to such treatment, such as proteins the size of collagen, lurks in the background.
It is accordingly desirable to provide a simplified oligopeptide structure having collagen-like properties for similar clinical applications, which can be readily manufactured in commercial quantities to obtain a substantially pure product, free of contaminants potentially dangerous to the recipient.
2. Description of Related Art
a) Collagen-like substances
Studies on the primary structure of collagen performed more than 20 years ago (Treatise on Collagen, volume 1; Ramachandran, ed., Academic Press, London, pp. 441-526, 1967; Protein Chem. 25:243-352, 1971) revealed the presence of glycine (Gly) as every third residue in a peptide chain, and a high proportion of the imino acid residues proline (pro) and hydroxyproline (Hyp) as characteristic of natural collagen from various mammalian species, including man (Biochem. 5:3803, 1966; Biochem. 10:2076, 1971). The studies also demonstrated that collagen contains a large number of tripeptide sequences of the form of Pro-Hyp-Gly, Pro-X-Gly, or X-Hyp-Gly, where X is an amino acid residue other than Pro, Gly, or Hyp. Numerous subsequent sequence studies have shown that Hyp mainly occurs as Y in the collagen repeating unit (X-Y-Gly).sub.n. Hyp is synthesized in vivo during the post-translational modification of collagen by the enzymatic hydroxylation of Pro after its incorporation into the polypeptide sequence, a critical step in the synthesis of collagen, since unhydroxylated collagen is not efficiently secreted from cells, has a lower denaturation temperature than fully hydroxylated collagen, and is therefore susceptible to nonspecific proteolysis. An examination of the primary structure of the .alpha.-chains of mammalian collagen reports that in triads of the type Gly-Pro-X and X-Pro-Gly, only the latter are hydroxylated (Biochemistry of Collagen, Ramachandran et al. eds, Plenum Press, New York, N.Y. p. 1, 1976). Further, polytripeptides in which X=Pro were the most efficient substrates for the hydroxylation, whereas increasing complexity of the side chain of other residues at X decreased the efficiency with which hydroxylation occurred (Biochem. 17:2892. 1978).
Early molecular models of collagen, comprising three peptide chains containing the above tripeptides, showed the chains twisted in a gradual right-handed helix, with each chain in the helix locked into a left-handed helix of poly-Pro II type by a large number of imino acid residues (Biochem. Biophys. Acta 109:314, 1965). In attempts to clarify the correlation between the primary and secondary structures of collagen, a variety of polypeptides with repeating sequences (Pro-Pro-Gly), (Pro-Hyp-Gly), and others have been synthesized and evaluated during the past twenty years. It was found that those sequences form highly stable triple-helical structures isomorphic to those of natural collagen, both in the solid state (J. Mol. Biol. 65:371, 1972). Studies have proposed that Hyp considerably stabilizes the collagen triple helix and contributes to the tertiary and quaternary structures of the protein by participating in intra- and intermolecular hydrogen bonding (Biochem. Biophys. Acta 322:166, 1972; Curr. Sci. 44:1, 1975).
Pioneering work on synthetic collagen models has also been done with polydisperse mixtures of sequential polytripeptides containing Pro and Gly (J. Mol. Biol. 43:461, 1969). Monodisperse oligotripeptides of (Pro-Pro-Gly).sub.n or (Pro-Hyp-Gly).sub.n sequences, where n=2-10, have also been prepared, with X-ray diffraction patterns of the former, wherein n was 4 or greater showing collagen-like diffraction patterns. Circular dichroism spectra of penta- and octadecapeptide films cast from solution are consistent with the conformation of collagen (Biopolymers 17:1215, 1978).
Notwithstanding the above described studies on the physical and biochemical characteristics of collagen and related proteins as well as numerous additional studies, little has been published firmly correlating these findings with properties of collagen which makes this protein clinically useful. One such property is that a great variety of cells, including epithelial cells, fibroblasts, platelets, and keratinocytes adhere to and migrate on specific regions within the triple-helical domain of types I, III and IV collagen; it has been recently reported (J. Biol. Chem. 265:14153, 1993) that triple helicity, more than the primary structure of the collagen peptide chains, is required for cell adhesion to and spreading on a specific collagen sequence, as the tested cells spread upon and adhered to the helical structure, while the single-stranded corresponding sequence was inactive in this regard (Biopolymers 33:1695, 1993).
Recently, it has been shown that bringing three (Pro-Hyp-Gly), chains in register by means of a template can significantly improve stability of the triple helix. The template approach has been reported previously, where the templates utilized are either 1,2,3-propanetricarboxylic acid (Biopolymers 18:2359, 1979), a lysine-lysine dimer (J. Theor. Biol. 153:585, 1991; Biopolymers 33: 1695-1707, Letters 334:272, 1993; Biopolymers 27:157, 1988; Biopolymers 19:1909, 1980; FEBS Letters 334:272, 1993; Biopolymers 25:1081, 1986) or Kemp triacid (cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid or KTA) (J. Am. Chem. Soc. 118:5156, 1996).
Each of the above-cited publications is incorporated herein by reference.
b) Polyethylene glycol
Polyethylene glycol (PEG) has been used as a protein modifier for the past twenty-five years. It has been used to react with free amino groups on proteins, using various activated PEGs, such as activated monomethoxypolyethylene glycol. Covalently linked to clinically useful proteins, it can improve their performance and safeness for therapeutic use. It was shown to decrease antigenicity of bovine serum albumin (BSA) (J. Biol. Chem. 252:3578, 1977) and to improve plasma half-life of the native protein by increasing its resistance to proteolytic enzymes (J. Biol. Chem. 252:3578, 1977). It also increases hydrophilicity at the surface of the modified protein (Biotechnol. Appl. Biochem. 17:115, 1993). PEG is recognized as safe for subcutaneous and oral uses in humans and in animals.